Conjugation systems for ubiquitin and Ubiquitin-like proteins (Ubls) are generally organized into enzyme cascades involving E1, E2, and, E3 enzymes. Among the several classes of ubiquitin E3s, the HECT E3s are mechanistically unique in that they form a covalent enzyme-ubiquitin intermediate and participate directly in the chemistry of protein ubiquitination. HECT E3s play important roles in several disease states (cervical cancer, Angelman syndrome, Liddle's syndrome), disease-related pathways (TGF-beta signaling, Notch signaling, trafficking of membrane proteins), and are commandeered by several types of viruses (human papillomaviruses, EBV, retroviruses, Ebola). In addition, a single HECT E3, HercS, is critical for conjugation of ISG15, a type 1 interferon-induced Ubl that plays a role in the innate immune response to viral and microbial infections. These examples highlight the importance of understanding the mechanism, function, and regulation of HECT E3s. We have made important contributions toward understanding HECT E3 functions and mechanisms and propose to extend recent discoveries through three major lines of investigation. The first line of investigation, driven by our investigations on yeast Rsp5, will focus on the regulation of Rsp5 and its human homologs by physically associated deubiquitinating enzymes (DUBs). This represents a novel form of E3 regulation that may represent a point of intervention in altering HECT E3 function in human disease. The second line of investigation will address the mechanistic basis for the distinction between HECT E3s that catalyze K63-linked polyubiquitination and those that catalyze K48- linked polyubiquitination. This is an important problem because the ultimate consequences of these two types of ubiquitination are distinct. The third line of investigation will focus on the mechanism and regulation of ISG15 conjugation system by the HercS HECT E3. We will identify the complete set of factors required for HercS-dependent ISG15 conjugation and determine how this single E3 directs conjugation to perhaps hundreds of ISG15 target proteins. A thorough understanding of the biochemistry of ISG15 conjugation is essential for understanding the function of ISG15 in innate immune responses. Public health relevance: Protein modification by ubiquitin and Ubls serves to modify the stability, activity, or localization of many key cell regulatory proteins, and disruptions or alterations in these processes are seen in many disease states, particularly cancer. The elucidation of the biochemistry of the HECT family of ubiquitin ligases will impact our understanding of mechanisms of carcinogenesis, signaling pathways, and the response to viral and microbial infections.